Abstract
Genetic studies of Streptococcus mutans have benefited greatly from the numerous techniques that have been successfully adapted for use in this organism. One notable exception is the lack of a negative selection system that can be employed for the easy isolation of markerless in-frame deletions. In this study, we report the development of a galK/galactose-based negative selection system in S. mutans for this purpose. This system consists of a recipient strain (IFD140) that contains a deletion in the galKTE operon and a suicide vector (pIFD-Sm) that carries the S. mutans galK open reading frame fused to the constitutive lactate dehydrogenase (ldh) promoter. Using this system we created a markerless in-frame deletion in the β-galactosidase (lacG) gene within the S. mutans lactose operon. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of the galactose-resistant colonies and resulted in in-frame deletions in 50% of the screened isolates. Based on the ratio of galactose-resistant cells to total cells, we determined that plasmid excision occurred at a frequency of approximately 1/3000 cells. Furthermore, the simplicity of this system should make it adaptable for use in numerous other gram-positive and gram-negative organisms.
Original language | English (US) |
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Pages (from-to) | 95-102 |
Number of pages | 8 |
Journal | Oral microbiology and immunology |
Volume | 22 |
Issue number | 2 |
DOIs | |
State | Published - Apr 2007 |
Externally published | Yes |
Keywords
- Galactose
- In-frame deletion
- Negative selection
- Streptococcus mutans
- galK
ASJC Scopus subject areas
- Microbiology
- Immunology
- General Dentistry
- Microbiology (medical)